Methods for specific detection of nucleic acid sequences using in vitro transcription and in situ sequencing

Abstract

The present disclosure relates, in general, to methods and compositions for detecting one or more nucleic acid sequences of interest (e.g., nucleic acid barcodes) in a biological sample in situ.

What is claimed is: 
     
         1 . A method of determining the presence and/or localization of a nucleic acid sequence of interest in one or more fixed mammalian cells within a biological sample in situ, the method comprising:
 (a) reacting, within the one or more fixed mammalian cells, (i) a DNA molecule comprising the nucleic acid sequence of interest operably linked to a sequence-specific RNA polymerase promoter, and (ii) a sequence-specific RNA polymerase to generate an RNA transcript of the nucleic acid sequence of interest;   (b) reacting the RNA transcript in situ with a reverse transcriptase enzyme to generate a cDNA molecule comprising the nucleic acid sequence of interest; and   (c) in situ sequencing the cDNA molecule to visualize the nucleic acid sequence of interest in the one or more fixed mammalian cells thereby to determine the presence and/or localization of the nucleic acid sequence of interest.   
     
     
         2 . The method of  claim 1 , wherein the method comprises, prior to step (a), contacting the fixed mammalian cell with an RNase to degrade endogenous RNA molecules. 
     
     
         3 . The method of  claim 1 , wherein the DNA molecule is, or is derived from, an exogenous nucleic acid molecule that is introduced to the one or more mammalian cells prior to fixation. 
     
     
         4 . The method of  claim 3 , wherein the nucleic acid sequence of interest is a barcode polynucleotide. 
     
     
         5 . The method of  claim 3 , wherein the exogenous nucleic acid molecule is incorporated into a genome of the one or more mammalian cells by viral transduction, site-specific nucleases, or site-specific recombinases. 
     
     
         6 . The method of  claim 5 , wherein the exogenous DNA molecule is introduced to the one or more mammalian cells using a viral vector selected from a lentiviral vector, a retroviral vector, an adenovirus vector, an HSV vector, a baculovirus vector, a virus-like particle, a pseudotyped virus-like capsid, an oncolytic viral vector, or an AAV vector. 
     
     
         7 . The method of  claim 1 , wherein the nucleic acid sequence of interest is an endogenous nucleic acid sequence and the promoter is an exogenous promoter. 
     
     
         8 . The method of  claim 1 , wherein the DNA molecule is generated by reverse transcribing with a DNA primer that hybridizes to a target RNA comprising the nucleic acid sequence of interest in the one or more fixed mammalian cells, wherein the DNA primer comprises:
 (a) a 5′ nucleic acid sequence comprising a sequence-specific RNA polymerase promoter, and   (b) a 3′ nucleic acid sequence that is complementary to a portion of the target RNA flanking the nucleic acid sequence of interest.   
     
     
         9 . The method of  claim 8 , wherein the method further comprises converting the DNA molecule to double-stranded DNA by second-strand synthesis. 
     
     
         10 . The method of  claim 8 , wherein the 5′ nucleic acid sequence of the DNA primer comprising the sequence-specific RNA polymerase promoter is dsDNA. 
     
     
         11 . The method of  claim 8 , wherein the target RNA molecule is wholly or partially digested following synthesis of the DNA molecule. 
     
     
         12 . The method of  claim 1 , wherein the in situ sequencing is sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-avidity. 
     
     
         13 . The method of  claim 1 , wherein:
 (a) the sequence-specific RNA polymerase promoter is a phage promoter, or a transcriptionally active variant thereof, and the sequence-specific RNA polymerase is a phage RNA polymerase;   (b) sequence-specific RNA polymerase promoter is a bacterial promoter and the sequence-specific RNA polymerase is a bacterial RNA polymerase;   (c) the sequence-specific RNA polymerase promoter is a eukaryotic promoter and the sequence-specific RNA polymerase is a eukaryotic RNA polymerase;   (d) the sequence-specific RNA polymerase promoter is a viral promoter and the sequence-specific RNA polymerase is a viral RNA polymerase; or   (e) the sequence-specific RNA polymerase promoter is a synthetic promoter and the sequence-specific RNA polymerase is a synthetic RNA polymerase.   
     
     
         14 . The method of  claim 1 , wherein the sequence-specific RNA polymerase promoter and the sequence-specific RNA polymerase are selected from the group consisting of:
 (i) a T7 promoter, or a transcriptionally active variant thereof, and a T7 RNA polymerase, respectively;   (ii) a T3 promoter, or a transcriptionally active variant thereof, and a T3 RNA polymerase, respectively; and   (iii) an SP6 promoter, or a transcriptionally active variant thereof, and an SP6 RNA polymerase, respectively.   
     
     
         15 . The method of  claim 1 , wherein the DNA molecule further comprises a transcriptional terminator. 
     
     
         16 . The method of  claim 1 , wherein the biological sample is fixed using a solution comprising formaldehyde and/or paraformaldehyde, by cryofixation, using a solution comprising an alcohol, or using a solution comprising glutaraldehyde. 
     
     
         17 . The method of  claim 1 , wherein the biological sample comprises a formalin-fixed, paraffin-embedded (FFPE) sample comprising the one or more mammalian cells. 
     
     
         18 . The method of  claim 1 , wherein the DNA molecule further comprises one or more polynucleotide sequences encoding exogenous proteins, endogenous proteins, or a mixture of exogenous and endogenous proteins. 
     
     
         19 . The method of  claim 18 , wherein the exogenous proteins are independently selected from the group consisting of a chimeric antigen receptor (CAR), an antibody, a T-cell receptor, a cytokine, a cell-surface receptor, a transcription factor, a signaling protein, and a protease. 
     
     
         20 . The method of  claim 1 , wherein the DNA molecule further comprises:
 (a) a polynucleotide sequence encoding an endogenous protein;   (b) a polynucleotide sequence encoding an endogenous RNA;   (c) a polynucleotide sequence encoding an exogenous RNA;   (d) a nucleic acid sequence that alters expression, function, and/or sequence of one or more genes; and/or   (e) a polynucleotide sequence encoding a viral genome.   
     
     
         21 . The method of  claim 1 , wherein the DNA molecule further comprises a polynucleotide sequence encoding a nucleic acid sequence that alters expression, function, and/or sequence of one or more genes. 
     
     
         22 . The method of  claim 21 , wherein the nucleic acid sequence that alters expression, function, and/or sequence of one or more genes is selected from the group consisting of an sgRNA, a gRNA an shRNA, and an miRNA. 
     
     
         23 . The method of  claim 1 , wherein the DNA molecule comprises a second sequence-specific RNA polymerase promoter configured to drive transcription of a second nucleic acid sequence of interest in the presence of a second sequence-specific RNA polymerase. 
     
     
         24 . The method of  claim 23 , wherein the second nucleic acid sequence of interest is a second barcode polynucleotide. 
     
     
         25 . The method of  claim 23 , wherein the second sequence-specific RNA polymerase promoter and the second sequence-specific RNA polymerase are selected from the group consisting of:
 (i) a T7 promoter, or a transcriptionally active variant thereof, and a T7 RNA polymerase, respectively;   (ii) a T3 promoter, or a transcriptionally active variant thereof, and a T3 RNA polymerase, respectively;   (iii) a SP6 promoter, or a transcriptionally active variant thereof, and a SP6 RNA polymerase, respectively;   (iv) a bacterial promoter or a transcriptionally active variant thereof, and a bacterial RNA polymerase, respectively;   (v) a eukaryotic promoter or a transcriptionally active variant thereof, and a eukaryotic RNA polymerase, respectively;   (vi) a viral promoter or a transcriptionally active variant thereof, and a viral RNA polymerase, respectively; or   (vii) a synthetic promoter and a synthetic RNA polymerase, respectively.   
     
     
         26 . The method of  claim 1 , wherein the DNA molecule further comprises a first padlock-binding sequence and a second padlock-binding sequence, wherein said first and second padlock-binding sequences flank a region comprising the nucleic acid sequence of interest. 
     
     
         27 . The method of  claim 23 , wherein the DNA molecule further comprises a third padlock-binding sequence and a fourth padlock-binding sequence, wherein said third and fourth padlock-binding sequences flank a region comprising the second nucleic acid sequence of interest. 
     
     
         28 . The method of  claim 26 , wherein, prior to in situ sequencing, step (c) further comprises the steps of:
 (i) contacting the cDNA with a first padlock probe comprising a 5′ end and a 3′ end, wherein the first padlock probe comprises a 5′ nucleic acid sequence which is reverse complementary to the first padlock-binding site and a 3′ nucleic acid sequence which is reverse complementary to the second padlock-binding site, thereby allowing the 5′ and 3′ nucleic acid sequences to hybridize to the cDNA;   (ii) extending the 3′ end of the first padlock probe through the nucleic acid sequence of interest using a DNA polymerase;   (iii) ligating the 5′ end of the padlock probe to the extended 3′ end of the padlock probe, thereby generating a circular DNA template comprising a nucleic acid sequence reverse complementary to the nucleic acid sequence of interest; and   (iv) using rolling circle amplification of the DNA template to generate additional copies of the nucleic acid sequence of interest.   
     
     
         29 . The method of  claim 1 , wherein the in situ sequencing is performed directly on the cDNA.